Natural Killer-Based Therapy: A Prospective Thought for Cancer Treatment Related to Diversified Drug Delivery Pathways

Immunotherapy has been a research hotspot due to its low side effects, long-lasting efficacy, and wide anti-tumor spectrum. Recently, NK cell-based immunotherapy has gained broad attention for its unique immunological character of tumor identification and eradication and low risk of graft-versus-host disease and cytokine storm. With the cooperation of a drug delivery system (DDS), NK cells activate tumoricidal activity by adjusting the balance of the activating and inhibitory signals on their surface after drug-loaded DDS administration. Moreover, NK cells or NK-derived exosomes can also be applied as drug carriers for distinct modification to promote NK activation and exert anti-tumor effects. In this review, we first introduce the source and classification of NK cells and describe the common activating and inhibitory receptors on their surface. Then, we summarize the strategies for activating NK cells in vivo through various DDSs. Finally, the application prospects of NK cells in tumor immunotherapy are also discussed.


Introduction
Cancer has become an intractable problem affecting human health around the world; nevertheless, there has been no universal tumor treatment strategy up to now [1].Surgical resection, chemotherapy, radiotherapy, and immunotherapy are generally the common methods for treating cancer in clinical practice, and surgical resection is still the most effective way for eradicating tumors in the early stage [2][3][4][5].Unfortunately, due to the lack of surgical conditions and insufficient therapeutic effect, less than 25% of cancer patients can receive surgical resection in time worldwide [6,7].Although chemotherapy and radiotherapy are also supposed to directly inhibit tumor progression besides surgery, both of them result in poor patient compliance and may cause serious adverse effects like cardio cytotoxicity, alopecia myelotoxicity, neurotoxicity, and nephrotoxicity [8][9][10][11].Since the Nobel Prize in Physiology and Medicine was produced in the field of immunotherapy in 2018, immunotherapy has been attracting more and more attention among numerous therapies against various types of cancer owing to its enhanced anti-tumor efficacy, attenuated side effects, and long-term immunological memory for preventing tumor metastasis and recurrence [12][13][14][15].Tumor cells can be eliminated during immunotherapy through the activation of the immune system, subsequently playing a long-term "surveillance" role in patients' bodies [16].
Natural killer (NK) cells are a type of critical immunocytes for immunotherapy, which can directly recognize and kill tumor cells without prior stimulation of tumor antigens, and seldom cause side effects like inflammatory storm.Thus, NK cells are regarded as another hot research direction alongside T-cell therapy [17].NK cells are the third largest type of lymphocytes other than T cells and B cells, which were first discovered in 1975 by Herberman and Kiessling [18][19][20].NK cells mainly develop from lymphoid progenitors in bone marrow, while some secondary lymphoid organs, such as the tonsils, spleen, and lymph nodes, also show a close relation to the maturation of NK cells [21].Mature NK cells belong to large granular lymphocytes in morphology, which mainly distribute in peripheral blood, lymph nodes, the spleen, and bone marrow, accounting for 5 to 15% of the lymphocytes in human peripheral blood [22].There are many commonly used methods for identifying and characterizing NK cells.For example, the NK cells in human peripheral blood, tissues, and bone marrow could be determined by the lack of CD3 on their surface, but they express neural cell adhesion molecule (NCAM, also named as CD56), while the ones in circulatory system could be determined by the expression of natural cytotoxic triggering receptor 1 (NCR1, also named NKp46 or CD335) [23][24][25][26].NK cells can be further divided into CD56 bright CD16 dim− and CD56 dim CD16 + subsets according to their maturation status.The less mature subset CD56 bright CD16 dim− is the main producer of cytokines, while CD56 dim CD16 + (the more mature subset) mainly exerts cytotoxic effects [27][28][29].
NK cells simultaneously possess cytotoxic and immunomodulatory characteristics without major histocompatibility complex (MHC) restriction, monitoring and inhibiting tumor growth in various ways [17,30].NK cells regulate their functions (cytotoxicity, proliferation, and cytokine production) based on the stimulation balance between activating and inhibition receptors expressed on the surface of the cell membrane, subsequently activating the apoptotic pathway of tumor cells without prior sensitization [31].Cytokine secretion is an important modality for NK cells to kill tumor cells.NK cells are the major source of distinct cytokines, producing tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) with tumoricidal properties [21,32].TNF-α usually plays an essential role in tumor treatment, which can directly kill tumor cells without significant toxicity to normal cells.TNF-α shows its cytotoxicity, antiviral property and immune regulating functions by binding to the tumor necrosis factor receptor (TNFR) on target cells, enhancing the host's immune function or acting with tumor vascular endothelial cells for vascular dysfunction [33][34][35].IFN-γ is another critical cytokine during tumor therapy, which can induce tumor cell apoptosis through the IFN-γ/STAT1 pathway, and the activation of STAT1 also inhibits tumor growth and establishes a dormant state [36].In addition, NK cells release cytotoxic substances like perforin and granzyme, eradicating tumor cells through an antibody-dependent cell-mediated cytotoxicity (ADCC) effect [37,38].
To enhance drug bioavailability and reduce side effects, drug delivery systems (DDSs) have been widely used in chemotherapy delivery.DDSs typically consist of two parts: drugs and their carriers.These systems can be categorized into two types: active targeting DDSs and passive targeting DDSs.The active targeting DDSs are usually modified with antibodies, proteins, nucleic acids, etc., which can bind to specific receptors or antigens at the target site, thus increasing the drug concentration at the target sites [39,40].In contrast, passive targeting DDSs are taken up by the target site through mechanisms such as capillary interception, intracellular endocytosis, cell membrane fusion, adsorption, and enhanced osmotic retention [41,42].Adopting DDSs can stimulate the activation of NK cells in vivo to exert their anti-tumor activity, while the NK cells can also be directly applied as tumor-targeted drug delivery carriers with the previous load of antibodies or DDS in vitro for enhanced tumor therapy.The anti-tumor property of NK cells can be activated by DDSs in various ways.For example, DDSs can enhance the activation signal of NK cells by stimulating the expression of NK-activating receptors or upregulating their ligands on tumor cells, or by blocking the expression of inhibitory receptors and their ligands [43][44][45].In addition, there are a large number of modifiable groups on the surface of NK cells and NK-derived exosomes (NEOs), and the endocytosis ability possessed by NK cells also enables them to be directly applied as drug delivery carriers for loading DDSs and exerting anti-tumor effects [46,47].In this review, we aimed to initially introduce both the stimulatory and inhibitory receptors expressed on the surface of NK cells, then describe the application of DDSs to activate NK cells via controlling the balance between stimulatory and inhibitory signals during tumor immunotherapy.Finally, we summarize the strategies of adopting NK cells and NEOs as drug delivery vehicles for anti-tumor therapy.

Receptors Expressed on NK Cells
Generally, the activation of T cells for tumor eradication can be achieved after their recognition of the antigens presented by MHC molecules on the surface of antigen-presenting cells (APCs) [48,49].In comparison, the activation or inhibition of NK cells is determined by the balance between both stimulatory and inhibitory signals on their surface without MHC restriction [31,50].When the activation signal is stronger than the inhibition one, the NK cells will be activated and later kill the target cells; otherwise, these NK cells will remain silent (Figure 1) [51].As tumor cells generally lack or even do not express MHC-I to escape from being recognized by T cells, NK cells exhibit outstanding application prospects for tumor immunity [52].NK cells can recognize tumor cells without high MHC-I expression, making up for the deficiency of T cells, and play an important role in cancer treatment [53].
ligands [43][44][45].In addition, there are a large number of modifiable groups on the surface of NK cells and NK-derived exosomes (NEOs), and the endocytosis ability possessed by NK cells also enables them to be directly applied as drug delivery carriers for loading DDSs and exerting anti-tumor effects [46,47].In this review, we aimed to initially introduce both the stimulatory and inhibitory receptors expressed on the surface of NK cells, then describe the application of DDSs to activate NK cells via controlling the balance between stimulatory and inhibitory signals during tumor immunotherapy.Finally, we summarize the strategies of adopting NK cells and NEOs as drug delivery vehicles for antitumor therapy.

Receptors Expressed on NK Cells
Generally, the activation of T cells for tumor eradication can be achieved after their recognition of the antigens presented by MHC molecules on the surface of antigen-presenting cells (APCs) [48,49].In comparison, the activation or inhibition of NK cells is determined by the balance between both stimulatory and inhibitory signals on their surface without MHC restriction [31,50].When the activation signal is stronger than the inhibition one, the NK cells will be activated and later kill the target cells; otherwise, these NK cells will remain silent (Figure 1) [51].As tumor cells generally lack or even do not express MHC-I to escape from being recognized by T cells, NK cells exhibit outstanding application prospects for tumor immunity [52].NK cells can recognize tumor cells without high MHC-I expression, making up for the deficiency of T cells, and play an important role in cancer treatment [53].

Activating Receptors on NK Cells
There are numerous types of activating receptors expressed on the surface of NK cells, which are responsible for NK triggering when they are interacting with the specific ligands expressed under stress, viral infection, or transformed cells [17].NKG2D is the most typical activating receptor on NK cells with the ability to recognize the structural analogs of MHC-I like ULBP and MICA/B, which can be upregulated through infection, stress, and tumor cells, and plays a critical role in processing tumor immune surveillance [54].CD16 is another important activating receptor expressed by NK cells.The Fab segment of the IgG antibody binds to the epitope of virus-infected or tumor cells, while the Fc segment of the IgG antibody binds to the CD16 on the NK cell surface, inducing NK cells to directly kill target cells [55,56].Cytotoxicity receptors (NCRs, including NKp46, NKp30, and NKp44) also play a core role in activating NK cells.NKp30 can bind to B7-H6 and BAG6 on tumor cells to trigger NK activation [57], and NKp46 is a 46 kDa glycoprotein composed of two C2-type extracellular Ig-like domains for NK activation, cytotoxic activity, and cytokine production by inducing protein tyrosine kinase-dependent signaling pathways [58].Both NKp30 and NKp46 promote NK activation by interacting with ITAM-containing adapter proteins, while NKp44 activates NK cells by interacting with Dap12 [59,60].
In addition, NK cells possess distinct cytokine receptors like interleukin 2 receptor (IL-2R), interleukin 12 receptor (IL-12R), interleukin 15 receptor (IL-15R), interleukin 18 receptor (IL-18R), and interleukin 21 receptor (IL-21R) on their surface.Applying cytokines for promoting both the activation and the proliferation of NK cells is regarded as one of the most common strategies to enhance NK cells' tumoricidal ability [61,62].For example, interleukin 2 (IL-2) is the first cytokine for NK activation in clinical practice, which has been approved by the FDA for treating metastatic kidney cancer and melanoma [63].Exogenous cytokine IL-2 not only induces continuous NK expansion and activation, but also enhances the maturation and persistence of isolated NK cells.IL-12 can stimulate the expression of chemokine (C-X-C motif) ligand 6 (CXCR6) and CD49a on NK cells in human peripheral blood, enhancing the expression of IFN-γ and NKG2C, but the use of IL-2 for cancer treatment may cause severe systemic toxicities [64].Interleukin 15 (IL-15) amplifies the NK population and increases the expression of NK activating-receptors without inducing the expansion of Treg populations [65].
Other common NK-activating receptors include DNAM-1, NKG2C, NKG2E, NKG2H, killer cell immunoglobulin-like receptors (KIRs, including KIR-2DS and KIR-3DS), etc. [66,67].After recognizing the ligands (CD155 and CD112), DNAM-1 not only promotes NKmediated clearance of transformed and virus-infected cells, but also plays a key role in the expansion and maintenance of virus-specific memory NK cells [68].In summary, upregulating the expression of NK-activating receptors or enhancing the binding to their ligands can facilitate NK activation.

Inhibitory Receptors on NK Cells
NK cells also express a variety of inhibitory receptors on their surface to counteract stimulatory signals produced from the activating receptors [69].Due to the fact that the activation state of NK cells is determined by the balance between stimulating and inhibitory signals, reducing the binding between inhibitory receptors to their ligands or inhibiting the expression of inhibitory receptors or their ligands can also effectively stimulate the anti-tumor efficacy of NK cells.Natural Killer Group 2A (NKG2A), a member of the C-type lectin-like NKG2 receptor family, is an important inhibitory receptor expressed by NK cells, accounting for about 20~80% of all receptors on healthy NK cells, and prevents them from accidentally injuring normal cells [70][71][72].Programmed death 1 (PD-1) is also a surface inhibitory receptor on NK cells [73].PD-1 blockage can enhance the eradication of NK cells to multiple myeloma cells and improve the release of IFN-γ from NK cells [74].T-cell immunoglobulin and mucin domain-containing protein-3 (Tim-3) is another type of inhibitory receptor on NK cells, and the activation of NK cells can be inhibited after binding phosphatidylserine (PS) with Tim-3 [75].

Drug Delivery System-Based NK Activation for Tumor Immunotherapy
Compared with T cells that need to be stimulated by tumor antigens presented by APC, the non-restricted killing of NK cells has attracted more and more attention in tumor immunotherapy.Nevertheless, some shortcomings of NK cells, such as insufficient invasion, biological activity, and immune surveillance, may diminish their tumor eradication ability to a certain extent.NK cells can be activated either by the balance of activating or inhibitory receptors on their surface, or by the action of ADCC to kill tumors.As is shown in Table 1, the most commonly adopted method for in vivo NK activation through DDS can be summarized as enhancing the activation signals and reducing the inhibitory signals of NK cells.IL-18R MVA-CD40L Activating the secretion of IL-18, IFN-α, etc. [ IL-6R and IL-12R PRINT-CpG Activating the secretion of IFN-γ, IL-6, IL-12, etc.

Ligand Expression Stimulation of Activating Receptors
Regulating the expression of activating receptors and their ligands through DDSs can destroy the transduction balance between stimulus and inhibition signals, overcome the immunosuppressive state, and activate the anti-tumor property of NK cells [44].Shi et al. designed a core-shell-structured and Zn 2+ -doped Zn-CoFe 2 O 4 @Zn-MnFe 2 O 4 superparamagnetic NP (ZCMF) to treat hepatoma [43].ZCMF showed highly controllable magnetic hyperthermia performance and ensured synergistic anti-tumor effects with mild magnetothermal therapy (MHT).As the mononuclear phagocyte system in the liver tends to capture intravenous-injected magnetic NPs, ZCMF could be abundantly accumulated in the liver after administration.Through MHT treatment, ZCMF significantly increased the expression of UL16-binding proteins (ULBPs) on the surface of HepG2 cells (human hepatoma cells).ULBPs are ligands for NKG2D, and the upregulation of ULBPs stimulates the activation of NK cells.According to the experimental results, the growth of both xenograft and orthotopic liver tumors was almost completely eradicated by inducing in vivo NK-related anti-tumor immunity.
Huang et al. uniformly combined polymer-coated carbon nanodots into an ordered mesoporous silica framework to obtain CD@MSN [90].CD@MSN was a type of biodegradable DDS for photothermal therapy.After CD@MSN treatment, the expression of activating receptors (NKG2D and NKp46) on NK cells obviously increased compared with a mesoporous silica NP (MSN) group.In addition, NK activation-related proteins, NKG2D ligands (including H60 and mrn41 proteins) on 4T1 cells (murine breast cancer cells), were also increased after NIR irradiation, hence stimulating the proliferation and activation of NK cells in vivo.
Another study has demonstrated that ruthenium (Ru)-based metal complexes possess outstanding tumor treatment efficacy and satisfy biosafety requirements.A Ru polypyridine complex (RuPOP) could regulate NK activation-related proteins on tumor cells to enhance the killing ability of NK cells against tumors.Based on this point, Xu et al. synthesized this DDS to enhance the expression of various ligands (MICA/B, ULBP1, ULBP2, ULBP4) for further NKG2D activation on MDA-MB-231 cells (human breast cancer cells).After therapy, the interaction between NK cells and tumor cells was strengthened [89].

Cytokine-Related Application
NK cells generally express distinct cytokine receptors (IL-21R, IL-2R, IL-12R, IL-15R, IL-18R, etc.) on their surface, and some pro-inflammatory cytokines like IL-2 and IL-15 can directly bind to their related receptors on NK cells for stimulation [96,97].Activating NK cells through cytokines is one of the relatively mature methods to strengthen the anti-tumor characteristic of NK cells [62].Cytokine-based NK stimulation has also been used in clinics or has reached the clinical trial phase, such as the application of Bempegaldesleukin (also named BEMPEG or NKTR-214, an IL-2 pathway agonist) for treating melanoma [98], and the short-term continuous infusion of IL-15 (NK expansion) for treating lymphoma [99].
Direct cytokine application: Cytokines with NK activation effects can be directly loaded into DDSs for precise tumor delivery and further activate NK cells.IL-2 is an important cytokine regulating the survival, proliferation, and differentiation of activated NK cells, which can directly promote NK activation after administration.Zhang et al. from the Huazhong University of Science and Technology constructed a TME-sensitive red cell membrane-coated nanogel (NR) containing both paclitaxel and IL-2 (NR P+I ) to treat tumors with the synergism of chemotherapy and immunotherapy (Figure 2) [78].As IL-2 could promote the proliferation and activation of NK cells, the absolute amount of NK cells (CD3 − NK1.1 + ) in murine melanoma tumors (B16-F10 cells) after treatment with NR P+I was 1.6 and 1.5 times higher than PTX + IL-2 and NR I groups, respectively.Zhang from Shandong University developed two types of micelles exhibiting both pH and redox sensitivity, namely IL-15-loaded micelles (IL-15-NPs) and Trp2/CpG-coloaded micelles (Trp2/CpG-NPs), which enhanced the tumoricidal effect of NK cells after interacting with IL-15-NPs, and promoted T-cell activation through DC antigen presentation and macrophage repolarization through Trp2/CpG-NPs, respectively [80].The experimental results demonstrated that after treatment with IL-15-NPs, the infiltration of NK cells in B16-F10 tumors increased obviously, and the tumor inhibition rate reached 93.76%.Meanwhile, there is also an increased infiltration of both T cells and NK cells in tumors.sensitivity, namely IL-15-loaded micelles (IL-15-NPs) and Trp2/CpG-coloaded micelles (Trp2/CpG-NPs), which enhanced the tumoricidal effect of NK cells after interacting with IL-15-NPs, and promoted T-cell activation through DC antigen presentation and macrophage repolarization through Trp2/CpG-NPs, respectively [80].The experimental results demonstrated that after treatment with IL-15-NPs, the infiltration of NK cells in B16-F10 tumors increased obviously, and the tumor inhibition rate reached 93.76%.Meanwhile, there is also an increased infiltration of both T cells and NK cells in tumors.Stimulation of cytokine secretion: Some DDSs can stimulate the body to secrete various cytokines for inducing NK activation.Perry et al. designed particle replication in nonwetting templates (PRINT) NPs as the nanoplatform to deliver CpG (potent Toll-like receptor TLR9 agonist) to mouse lungs via oral duct infusion [82].The combination of both CpG and TLR9 initiated a series of innate and acquired immune responses, which activated DC cells and subsequently promoted the secretion of pro-inflammatory cytokines (namely IL-6, IL-12, etc.) for NK activation.The results demonstrated that local delivery of PRINT-CpG to the lungs effectively promoted substantial tumor regression and limited systemic toxicity in two murine orthotopic metastasis models (344SQ cells: lung adenocarcinoma cells; KAL-LN2E1 cells: lung squamous carcinoma cells).Hubertus Hochrein's team applied recombinant modified vaccinia virus Ankara (rMVA)-encoded co-stimulating CD40L to treat solid tumors (mouse melanoma and lymphoma), inducing the secretion of cytokines like IL-18 and IFN-α after intravenous injection to ensure NK expansion and activation, eventually killing the tumor cells [81].In addition, Kuang et al. found that chiral NPs strengthened the tumoricidal ability of NK cells through stimulating cytokine (IL-18 and IL-12) expression [83].The results exhibited that the mice treated with L-type NPs induced (33.62 ± 3.41)% NK activation in tumors, which was 1.39 times higher than D-type NP groups.

Expression or Binding Prevention of Inhibitory Receptors' Ligands
The stimulus-inhibitory signal transduction balance of NK cells can be effectively broken through the prevention of the NK-expressed inhibitory receptors from binding to their ligands.Meanwhile, the immunosuppressive state of NK cells can be overcome, and their anti-tumor property will be subsequently enhanced.
Blocking the binding between human leukocyte antigen-E (HLA-E) and NKG2A is beneficial to enhance NK-mediated immunotherapy against tumors.Recently, studies have shown that both inorganic and organic selenite are available to down-regulate the expression of HLA-E in tumor cells but activate NK cells.Yang et al. prepared a type of enzyme and reactive oxygen species (ROS) double-sensitive selenopeptide NP (SeP/DOX) [87].After intravenous injection, the NPs precisely aggregated in tumors by recognizing the overexpressed αvβ3 integrin on endothelial cells in the neovascular system of MDA-MB-231 tumors.The selenopeptide in NPs was decomposed in the high-ROS TME, and the oxidative metabolite alkylselenic acid activated the immune response of NK cells by blocking the binding of HLA-E with NKG2A.Another selenium compound for NK activation was designed by Xu et al., who prepared a cytosine diselenide compound (Cyt-SeSe-Cyt) and diselenide-pemetrexed (Pem/Se) [45].The diselenide bond in this compound broke under low-dose γ-ray irradiation to produce selenite acid, which not only inhibited the expression of HLA-E on breast cancer cells, but also blocked the NK-expressed inhibitory immune checkpoint to promote tumor eradication through NK cells.In addition, Xu and his teammates prepared PSeR/DOX NPs through the self-assembly of amphipathic polymer PSeR containing selenium and DOX [88].In vitro experiments showed that the selenite molecules produced by radiation therapy significantly improved the anti-tumor activity of NK cells.

ADCC Induction of NK Cells
When the Fc receptors on tumor cells bind to the Fab fragments from monoclonal antibodies (lgG), the CD16 on NK cells could identify the Fc domains of lgG and kill the tumor, which is also known as the ADCC effect [100].Generally, the NK-mediated ADCC effect will be largely limited by the heterogeneity of tumor antigens.This is attributed to the fact that it is difficult to identify a specific tumor antigen stably expressed on various tumors, and only specific antigens can be recognized through antibody-mediated ADCC.Thus, it is urgent to seek a suitable route to strengthen the NK-induced ADCC effect for tumor therapy.
To overcome the aforementioned limitation, Ji et al. developed a strategy that did not require the presence of specific antigens on distinct tumors (B16-F10 cells; 4T1 cells; MDA-MB-231 cells; Raji: human lymphoma cell line) to ensure tumor targetability [76].After binding Fc fragments or therapeutic monoclonal antibodies to the N-terminal of the peptide, the pH-low insertion peptides (pHLIPs) underwent conformational transformation in the weakly acidic TME and then assembled to the membrane of tumor cells.These Fc fragments or antibodies bound to the NK-expressed CD16 to directly activate NK cells and initiate the ADCC effect for tumor eradication.Experimental results indicated that this strategy had the potential to treat various primary tumors and tumor metastases.Lu et al. designed an immunomodulatory NP (IMN) that can directly modify NK activation signals on tumor cells (B16F10 cells, 4T1 cells, and MDA-MB-231 cells) [77].After intravenous injection, the NPs could accumulate in tumors and decompose the glucose-modified poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) shell in the TME to release the bifunctional core (nBSA-PBA-IgG).nBSA-PBA-IgG attached to the tumor cell membrane through interacting between phenylboronic acid (PBA) and sialic acid and realized the in situ modification of NK-activating ligand (IgG) on tumor cells to activate the nearby NK cells.

Other Strategies for NK Cell Stimulation
In addition to the above methods, studies have revealed that phosphite-modified phosphorus dendritic macromolecules can also promote NK proliferation in peripheral blood monocytes (PBMCs) by inhibiting regulatory T cells (Tregs) and regulating the signals between monocytes and NK cells [101,102].Based on this strategy, Shi et al. constructed an immune-regulatory drug-carrying NP containing phosphorus crown macromolecules, M-G1-TBPNa@DOX [91].The experimental results showed that the inherent immunomodulatory activity of these NP micelles promoted the proliferation of NK cells in PBMCs, which were recruited to the tumor site through blood circulation, and exerted anti-tumor efficacy in coordination with other immune cells.
In addition, researchers have also synthesized bispecific T-cell conjugates (BiTEs) and tri-specific killer conjugates (TriKEs), which effectively enhance the cytotoxicity of NK cells against tumors.These structures are typically connected by a single-chain antibody fragment (scFv) of the anti-CD16 antibody with one BiKE or two TriKEs of tumor antigenspecific antibodies' scFv.These structures allow high-affinity CD16 + NK cells to bind to tumor cells, hence overcoming the limitations caused by receptor polymorphism.Felices et al. designed a TriKE of tri-specific biologic drugs containing distinct antibody fragments (including single-domain antibodies against CD16, single-chain antibodies against HER2 antigens on cancer cells, and IL-15), which could activate NK cells through different pathways [85].These antibody fragments could simultaneously bind with both NK cells and ovarian cancer cells, and the IL-15 portion was applied to specifically mediate IL-15 signaling on NK cells when inducing ADCC effects for triggering robust NK expansion.Compared with the control group, TriKEs promoted the proliferation of NK cells and ensured the highest NK amount among all experimental groups during the entirety of tumor therapy.
Studies have shown that NPs containing cationic polymers could promote the production of pro-inflammatory cytokines and induce a strong humoral response [103,104].Park et al. developed a kind of cationic NP (cNP) by applying polydopamine (PDA) chemistry to immobilize PEI onto the surface of magnetic NPs for NK activation (Figure 3) [95].cNP could increase the expression of chemokine receptors (CCR4 and CXCR4) on NK cells.In vitro experiments showed that the cytotoxicity of cNP-treated primary NK and NK-92MI cells against MDA-MB-231 cells was more than twice as high as that of untreated NK cells.Meanwhile, in vivo studies also proved that cNP-treated NK cells significantly inhibited tumor growth.

NK Cells and NK-Derived Exosomes as Drug Delivery Vehicles for Tumor Immunotherapy
NK cells possess numerous advantages in immunotherapy, including abundant cell sources [71,105], high safety profiles [106,107], reduced susceptibility to cytokine release syndrome (CRS), minimal neurotoxicity, and low immunostimulatory properties during therapy [108][109][110][111].Moreover, NK-based immunotherapies (such as CAR-NK) have recently demonstrated satisfactory clinical outcomes [112][113][114][115].However, the therapeutic effect of NK cells against solid tumors is usually inadequate due to insufficient local NK infiltration and NK deactivation induced by the TME [116,117].Meanwhile, natural NK cells cannot perform targeted cancer immunotherapy due to the lack of cell-specific receptors [118].In general, anti-tumor efficacy can be promoted with increased NK infiltration in tumors.Therefore, it is valuable to seek suitable methods to increase intratumoral NK recruitment and infiltration.
Fortunately, the emergence of DDSs provides a novel idea to solve the above problems.Recently, it has been feasible to design distinct NK-derived drug delivery platforms based on the existence of numerous modifiable groups on NK cell surfaces.According to the latest studies, NK cells have been successfully surface-modified with various antibodies and proteins [118,119].In this section, we will focus on the application of NK cells themselves and NK-derived exosomes as drug delivery vehicles for anti-tumor immunotherapy (Figure 4).

NK Cells and NK-Derived Exosomes as Drug Delivery Vehicles for Tumor Immunotherapy
NK cells possess numerous advantages in immunotherapy, including abundant cell sources [71,105], high safety profiles [106,107], reduced susceptibility to cytokine release syndrome (CRS), minimal neurotoxicity, and low immunostimulatory properties during therapy [108][109][110][111].Moreover, NK-based immunotherapies (such as CAR-NK) have recently demonstrated satisfactory clinical outcomes [112][113][114][115].However, the therapeutic effect of NK cells against solid tumors is usually inadequate due to insufficient local NK infiltration and NK deactivation induced by the TME [116,117].Meanwhile, natural NK cells cannot perform targeted cancer immunotherapy due to the lack of cell-specific receptors [118].In general, anti-tumor efficacy can be promoted with increased NK infiltration in tumors.Therefore, it is valuable to seek suitable methods to increase intratumoral NK recruitment and infiltration.
Fortunately, the emergence of DDSs provides a novel idea to solve the above problems.Recently, it has been feasible to design distinct NK-derived drug delivery platforms based on the existence of numerous modifiable groups on NK cell surfaces.According to the latest studies, NK cells have been successfully surface-modified with various antibodies and proteins [118,119].In this section, we will focus on the application of NK cells themselves and NK-derived exosomes as drug delivery vehicles for anti-tumor immunotherapy (Figure 4).In past studies, a large number of NK cells have been modified with antibodies and other proteins by modifying the surface of NK cells.For example, Yang et al. modified the synthesized CD30-specific aptamer on NK surfaces to obtain engineered NK cells (ApEn-NK) [118].ApEn-NKs were able to specifically bind to CD30-expressing lymphoma cells and had a greater capacity to kill lymphoma cells compared with NK cells by unmodified antibodies.Similarly, Li et al. modified IgG antibodies onto the glycocalyx on the NK surface to obtain Herceptin-NK-92MI, exhibiting enhanced activity in vitro, and induce the lysis and death of HER2 + cancer cells [119].The basic structure of a CAR includes an antigen recognition domain, an extracellular hinge domain, a transmembrane domain, and an intracellular signal domain composed of scFv [120].In past studies, a large number of NK cells have been modified with antibodies and other proteins by modifying the surface of NK cells.For example, Yang et al. modified th synthesized CD30-specific aptamer on NK surfaces to obtain engineered NK cells (ApEn NK) [118].ApEn-NKs were able to specifically bind to CD30-expressing lymphoma cell and had a greater capacity to kill lymphoma cells compared with NK cells by unmodified antibodies.Similarly, Li et al. modified IgG antibodies onto the glycocalyx on the NK sur face to obtain Herceptin-NK-92MI, exhibiting enhanced activity in vitro, and induce th lysis and death of HER2 + cancer cells [119].The basic structure of a CAR includes an anti gen recognition domain, an extracellular hinge domain, a transmembrane domain, and a intracellular signal domain composed of scFv [120].
CAR-NK cells are produced using genetic engineering technology, which enables th expression of CAR on NK cells [121].Specifically, genetic engineering technology involve integrating a chimeric antibody CAR onto NK cells, enabling them to recognize and attac tumor cells.The chimeric antibody CAR can enable CAR-NK to accurately and efficientl kill tumor cells in vivo.Compared with CAR-T cells, CAR-NK cells show a wider anti CAR-NK cells are produced using genetic engineering technology, which enables the expression of CAR on NK cells [121].Specifically, genetic engineering technology involves integrating a chimeric antibody CAR onto NK cells, enabling them to recognize and attack tumor cells.The chimeric antibody CAR can enable CAR-NK to accurately and efficiently kill tumor cells in vivo.Compared with CAR-T cells, CAR-NK cells show a wider anti-tumor spectrum and better clinical safety (as they never induce graft versus host disease) and are less prone to cytokine release syndrome [122,123].Recently, CAR-NK cells have been widely studied in clinical practice; for example, the China National Medical Products Administration approved the clinical trial application of IBR854 cell injection (CAR-NK injection derived from homologous peripheral blood) in 2022 for treating solid tumors.Exogenous cytokine IL-21 can not only induce continuous NK expansion and activation, but also enhances the maturation and persistence of isolated NK cells.However, low doses of IL-21 only produce weak proliferation and differentiation effects on NK cells, while overdose may cause severe toxicity effects like systemic cytokine storm, lymphocyte reduction, and an increase in transaminases.To circumvent the side effects of IL-21 during administration of the highest measure, Cai et al. implanted -N 3 /-BCN motifs on both NK and lymphoma cells by glycoconjugate engineering and coupled IL-21-containing NPs (ILNPs) on NK cells [124].The above two cells were modified with complementary bioorthogonal chemical moieties, which could achieve effective tumor recognition and promote NK infiltration into tumor tissue.Meanwhile, the above redox NPs coupled on NK cells could continuously release IL-21 in the TME, generating an effective and sustained "pseudo-autocrine" stimulation of NK cells.In short, this strategy provided a novel idea for promoting NK proliferation and activation, enhancing the therapeutic potential, and limiting the systemic toxicity during cancer therapy.Kim et al. prepared a kind of DOX-encapsulated pH-sensitive micelle, which was linked to the surface thiol groups on NK cells via maleimide-thiol coupling chemistry to obtain RE-NK cells [125].When the circulating NK cells approached tumor tissue together with chemokines, these NK cells initiated the formation of immune synapses (ISs), and subsequently released acidic granules to stimulate drug release.When RE-NK cells attacked tumors, the local acidic TME could realize rapid micelle disassembly and eventually ensured the sufficient release of DOX.
In addition to directly modifying NPs onto NK cells in vitro, researchers at Cornell University prepared a liposome carrying TRAIL protein, which was injected in vivo and subsequently immobilized on the NK cell surface after binding to the antibody against NK1.1 to obtain "super-natural killer cells" [126].These "super-natural killer cells" could accumulate in tumor-draining lymph nodes for a long time, inducing the apoptosis of cancer cells and preventing the lymphatic spread of subcutaneous colon cancer in mice.

NK Cells Loading Nanoparticles via Endocytosis
Living NK cells possess an active NP uptake ability; thus, the loading of nanoparticles can also be achieved by active cellular uptake [127].Based on this, Jiang et al. developed superparamagnetic iron oxide NPs (Fe 3 O 4 @PDA NPs) composed of magnetic Fe 3 O 4 cores with PDA shells for magnetically targeted therapy [128].Fe 3 O 4 @PDA NPs had excellent stability and biocompatibility in vivo, and they could be actively ingested by NK cells without affecting the biological function of NK cells.Both in vitro and in vivo studies demonstrated that Fe 3 O 4 @PDA NP-labeled NK cells possessed higher intratumoral aggregation with the presence of a magnetic field, which significantly inhibited the tumor growth through increasing the apoptosis of A549 cells (human non-small-cell lung cancer cells).Similarly, Cheng et al. obtained Cy5.5-conjugated Fe 3 O 4 /SiO 2 core/shell NPs, and co-incubated them with NK-92MI cells to enter the interior of these cells via endocytosis [129].The NP-loaded NK cells were then administrated to mice suffering from B cell lymphoma through tail vein injection, and the location of NK-92MI cells could be accurately controlled by a magnetic field.The results showed that the tumor penetration rate of NP-loaded NK-92MI was 17 times higher than that of normal NK cells under an external magnetic field, while the tumor-killing activity of NK cells was not affected.Fernandes et al. designed a magnetically responsive NK:IONP by modifying iron oxide nanoparticles (IONPs) onto NK cells (Figure 5) [130].These engineered NK cells could be precisely delivered to the desired target tumor site under magnetic guidance, eventually achieving maximum tumor eradication efficacy.

NK-Derived Exosomes as Drug Delivery Vehicle for Tumor Immunotherapy
NEOs possess similar immune regulatory functions to NK cells but have better tumor permeability compared with NK cells as they have a smaller particle size (40-150 nm), making them not easily affected by the TME [131].Clinical studies revealed that NEOs have a tumor-killing ability and can be applied as an anti-tumor agent alone [132].NEOs also have multitudinous advantages like high safety, easy storage and transportation, and

NK-Derived Exosomes Drug Delivery Vehicle for Tumor Immunotherapy
NEOs possess similar immune regulatory functions to NK cells but have better tumor permeability compared with NK cells as they have a smaller particle size (40-150 nm), making them not easily affected by the TME [131].Clinical studies revealed that NEOs have a tumor-killing ability and can be applied as an anti-tumor agent alone [132].NEOs also have multitudinous advantages like high safety, easy storage and transportation, and tumor targeting [133,134].However, the anti-tumor effect of NEOs is not strong enough when they are separately adopted [135].In order to better exert the anti-tumor ability of NEOs, their outstanding drug-loading capacity was utilized to improve their immune regulatory ability.Zhao et al. evaluated the application of extracellular vesicles derived from amplified NKs (eNK-EXO) in treating human ovarian epithelial cells (IOSE80 cells) [46].The results indicated that eNK-EXO expressed NK-characteristic proteins and cytotoxic substances, which could be selectively ingested by ovarian cancer cells, directly inducing cell apoptosis and enhancing the cytotoxicity of NK cells with impaired function in TME.Then, the anti-tumor drug cisplatin was loaded into eNK-EXO for preparing DDP.DDP exerted a critical effect on inhibiting the proliferation of ovarian cancer cells, especially chemotherapy-resistant cells, and significantly promoted cell apoptosis.
In addition to anti-tumor agents, Huang et al. loaded hydrophilic small interfering nucleic acids (siRNAs) into NEOs through electroporation, then loaded photosensitizer Ce6 onto the NEO membrane to construct light-activatable silencing NEOs (LASNEOs) (Figure 6) [135].After entering cells, LASNEOs could produce ROS under 660 nm light irradiation, which achieved satisfactory photodynamic therapy through promoting the gene-silencing effect of siRNA after its entry and release into the cytoplasm of HepG2-Luc cells.

Summary and Outlook
In this review, we first introduced the origin, classification, and anti-tumor mechanism of NK cells, then described the activation of NK cells by DDS in tumor immunotherapy.NK cells are a type of important tumor killer cell in vivo, and the activation state of NK is determined by the balance between activation and inhibition signals on the NK surface.Tumor cells can evade the recognition of T cells due to the lack of MHC-I expres-

Summary and Outlook
In review, we first introduced the origin, classification, and anti-tumor mechanism of NK cells, then described the activation of NK cells by DDS in tumor immunotherapy.NK cells are a type of important tumor killer cell in vivo, and the activation state of NK is determined by the balance between activation and inhibition signals on the NK surface.Tumor cells can evade the recognition of T cells due to the lack of MHC-I expression.In comparison, NK cells can directly induce the apoptosis of tumor cells without MHC restriction, which plays an important role in tumor immunity and exerts enhanced tumoricidal efficacy during treatment.NK cells are also an important source of cytokines in vivo, which can induce tumor eradication by secreting distinct cytokines (such as TNF-α, IFN-γ, IL-2, etc.).The surface of NK cells expresses a large number of signaling receptors, which can be finally classified into activating receptors (like NKG2D, KIR-2DS, NKp46, DNAM1, IL-2R, etc.) and inhibitory receptors (namely NKG2A, NKG2B, Tim-3, PD-1, etc.).With the diversity of DDSs, NK cells exert anti-tumor activity after being stimulated by regulating the balance between active and inhibitory signals of NK in vivo.NK cells can be activated through stimulating the expression of stimulatory receptors or their ligands, inhibiting the expression of NK inhibitory receptors or blocking the binding to their ligands, or loading the cytokines with NK activation properties into various DDSs to directly stimulate NK cells after administration.In addition, NK cells exhibit the advantages of easy culture and expansion in vitro, and can also be directly applied as drug delivery carriers.Modifiers or NPs are firstly loaded on NK cell surfaces to activate NK cells, and these activated NK cells can be later transfused back into the body to exert anti-tumor effects.NEOs, which possess anti-tumor efficacy and tumor targeting, are also used as drug delivery carriers, loading anti-tumor agents or RNA to synergistically exert NEOs' anti-tumor effect.
Immunotherapy based on NK activation has indeed achieved certain promising effects in treating hematologic tumors and has brought many expectations for curing tumors.Nevertheless, its broad application against tumors in clinicals has still been limited due to the following problems: (1) Due to the insufficient infiltration of NK cells in solid tumors and the influence of the TME on NK activity, the effect of NK-based immunotherapy against solid tumors is not ideal.(2) The half-life of NK cells is relatively short, and although NKbased therapy shows higher safety compared with T cells, NK-based immunotherapy requires more repeated administration to ensure treatment efficacy.(3) Due to the lack of cell-specific receptors, NK cells cannot directly target cancer sites.Although there have been some studies on modifying NK cells to make them more effective in tumor targeting, modified NK cells have not been involved in clinical studies in treating solid tumors.In brief, because of the above shortcomings of NK-based therapies, it remains necessary to seek more suitable solutions before further clinical application to cancer therapy.
Despite the fact that there are various problems that need urgently addressing in NKbased tumor therapy, with the progress of biomedical technology and the further research on anti-tumor treatment, we believe that these current obstacles in the development of immunotherapy based on NK activation can be overcome in the future.Meanwhile, the essentiality of NK therapy in tumor treatment will also be ultimately improved.

Figure 1 .
Figure 1.Schematic illustration of representative activating and inhibitory receptors and some ligands on the surface of NK cells.

Figure 1 .
Figure 1.Schematic illustration of representative activating and inhibitory receptors and some ligands on the surface of NK cells.
the expression of CCR4 and CXCR4 on NK[95]

Figure 2 .
Figure 2. Schematic illustration of the preparation and functional mechanism of NRP+I.After accumulating in tumors through the EPR effect, NRP+I was rapidly expanded in acidic TME and released PTX to induce CRT exposure on tumor cells.Subsequently, IL-2 was continuously released into the TME to activate CTL and NK cells.Picture reprinted from ref. [78].

Figure 2 .
Figure 2. Schematic illustration of the preparation and functional mechanism of NR P+I .After accumulating in tumors through the EPR effect, NR P+I was rapidly expanded in acidic TME and released PTX to induce CRT exposure on tumor cells.Subsequently, IL-2 was continuously released into the TME to activate CTL and NK cells.Picture reprinted from Ref. [78].Adoption of cytokine-encoded plasmids or mRNAs: In addition to the direct delivery of cytokines, plasmids or mRNAs encoded with cytokines can also be loaded by DDSs for NK activation.Gao et al. prepared RDMCM NPs (RDMCM/pIL-12 + PD-1/PD-L1i) composing two copolymers (cRGD-PEG-PCL and MPEG-PCL-MPEG) and DOTAP for loading an IL-12-encoded plasmid (pIL-12) and PD-1/PD-L1 small-molecule inhibitor (PD-1/PD-L1i) for melanoma (B16-F10 cells) therapy [79].After intravenous injection, the above dual drugs were accurately delivered to tumors through the targetability to integrin αvβ3 receptor, and the expressed IL-12 on tumor cells was upregulated through the cellular uptake of pIL-12.This phenomenon indicated that the amount of activated NK cells in the RDMCM/pIL-12 + PD-1/PD-L1i group was almost twice that of the control group.Dong et al. developed a type of lipid NP (DAL-LNPs) for encapsulating mRNAs encoding multiple cytokines (including IL-12, IL-27, and GM-CSF) for B16-F10 melanoma treatment [84].IL-12 activated both T cells and NK cells through the Stat4 pathway, while IL-27 could activate these cells through the Stat1 and Stat3 pathways.After intratumoral injection, the enhanced intratumoral infiltration of NK cells was achieved.

Figure 3 .
Figure 3. Schematic diagram of enhancing the anti-tumor effect of NK cells by cNPs.Natural NK cells cannot directly recognize tumor cells.After the stimulation of cNPs, the expression of CCR4 and CXCR4 on NK was induced and the ability of NK cells to recognize and kill tumor cells was increased.Picture reprinted from Ref [95].

Figure 3 .
Figure 3. Schematic diagram of enhancing the anti-tumor effect of NK cells by cNPs.Natural NK cells cannot directly recognize tumor cells.After the stimulation of cNPs, the expression of CCR4 and CXCR4 on NK was induced and the ability of NK cells to recognize and kill tumor cells was increased.Picture reprinted from Ref. [95].

4. 1 .
NK Cells as Drug Delivery Vehicle for Tumor Immunotherapy 4.1.1.NK Cell Loading Agent via Chemical Connection Due to the poor tumor tropism of NK cells, directly administrating innate NK cells can neither reach deep tumor sites nor exhibit tumor targetability.In order to allow NK cells to be tumor-targetable, some studies have loaded NPs or other modifiers onto the cell surface through the modifiable groups on NK cells to enhance NK cells' anti-tumor efficacy.

Pharmaceutics 2024 , 2 Figure 4 .
Figure 4. Schematic illustration of NK cells and NK-derived exosomes as drug delivery vehicles fo tumor immunotherapy.

Figure 4 .
Figure 4. Schematic illustration of NK cells and NK-derived exosomes as drug delivery vehicles for tumor immunotherapy.

Pharmaceutics 2024 , 21 Figure 5 .
Figure 5.The construction and application of NK:IONP for tumor therapy, including the isolation and surface modification of NK cells, the synthesis of NK:IONP, and the tumor targeting mechanism of NK:IONP.Picture reprinted from Ref [130].

Figure 5 .
Figure 5.The construction and application of NK:IONP for tumor therapy, including the isolation and surface modification of NK cells, the synthesis of NK:IONP, and the tumor targeting mechanism of NK:IONP.Picture reprinted from Ref. [130].

Figure 6 .
Figure 6.Schematic illustration of LASNEO-mediated collaborative tumor eradication.(A) Description of the preparation of siRNA and Ce6 dual-loaded LASNEO, and the photo-triggered LASNEO disassembly.(B) LASNEO collaboratively reprograms multiple types of immune cells for tumor immune stimulation.Picture reprinted from Ref. [135].

Table 1 .
Examples of various strategies for NK activation.